Tissue factor targeted thrombomodulin fusion proteins as anticoagulants

ABSTRACT

This invention relates to novel fusion proteins which are comprised of a targeting protein that binds tissue factor (TF), which is operably linked to the thrombomodulin (TM) EGF456 domain alone or in combination with at least one other TM domain selected from the group consisting of the N-terminal hydrophobic region domain, the EGF123 domain, the interdomain loop between EGF3 and EGF4, and the O-glycosylated Ser/Thr-rich domain, or analogs, fragments, derivatives or variants thereof. The fusion protein binds at the site of injury and prevents the initiation of thrombosis. The fusion protein can be used to treat a variety of thrombotic conditions including but not limited to deep vein thrombosis, disseminated intravascular coagulation, and acute coronary syndrome.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/376,566, filed May 1, 2002, which is incorporated herein in fullby reference.

BACKGROUND

Maintaining the proper balance between procoagulant and anticoagulantactivity within blood vessels is essential for normal hemostasis (Davie,E. W. et al. (1991) Biochemistry, 30(43):10363–10370). Perturbing thebalance toward coagulation leads to thrombosis, which can cause heartattack, stroke, pulmonary embolism, and venous thrombosis. There is aneed for more effective and safer anticoagulants for the treatment ofspecific thrombotic disorders.

Tissue factor (“TF”) is a transmembrane glycoprotein that is the majorinitiator of the coagulation cascade (Nemerson, Y. (1995) Thromb.Haemost. 74(1):180–184). Under normal physiological conditions active TFis not in contact with blood. During vascular injury, exposure to bloodof subendothelial TF and collagen leads to activation of coagulationfactors and platelets and subsequently to hemostatic plug formation. Theinappropriate induction of TF expression in a variety of clinicalsettings can lead to life threatening thrombosis and/or contribute topathological complications. TF exposure following plaque rupture isbelieved to be responsible for thrombotic occlusion leading to acutemyocardial infarction and stroke. In these settings, proinflammatorysignaling pathways activated by coagulation factors also contribute toedema formation and increased infarct size. Vascular injury associatedwith angioplasty leads to upregulation of TF on SMC's which is believedto induce cell signaling pathways associated with restenosis. TFoverexpression in cancer and gram-negative sepsis leads to lifethreatening thrombosis and activation of inflammatory pathways.

The factor VIIa (“FVIIa”)/TF complex is involved in the pathogenicmechanism in a variety of thrombotic diseases and the circulating levelof TF is a risk factor for certain patients. FVIIa and TF play uniqueroles in vascular injury in maintaining hemostasis and initiatingthrombosis. TF is expressed in the adventitia normally, but isupregulated and expressed inappropriately in the media and neointima invascular disease. TF expression in atherosclerotic plaques is increasedand shielded from the blood by a thin fibrous cap that may rupture toexpose TF. Surgical interventions such as balloon angioplasty, stenting,or endarterectomy damage the vessel wall and expose underlying TF. Inthe atherosclerotic, lipid-rich, thin-walled plaque, spontaneous ruptureor endothelial erosion leads to TF exposure and thrombosis, resulting inunstable angina and myocardial infarction. TF can circulate in cellderived microparticles and circulating TF levels are elevated inunstable angina suggesting that this circulating TF may contribute tothrombus formation (Soejima, H. et al. (1999) Circulation99(22):2908–2913). Often cancer is associated with a hypercoagulablestate attributed to overexpression of TF on tumor cells. Thispredisposes the patient to deep vein thrombosis, pulmonary embolism andlow grade disseminated intravascular coagulation (“DIC”). DIC results inmicrovascular fibrin deposition contributing to multi-organ failure.Results from acute arterial injury models of thrombosis indicate thatprotein based inhibitors of FVIIa/TF, such as active site inhibitedfactor VIIa (“FVIIai”) and tissue factor pathway inhibitor (“TFPI”), areeffective antithrombotics with less bleeding compared to thrombin andfactor Xa (“FXa”) inhibitors. In addition, FVIIa/TF inhibition issuperior to other anticoagulants (e.g., heparin, FXa inhibitors) inpreventing neointimal thickening and vascular stenosis following ballooninjury (Jang, Y. et al. (1995) Circulation 92(10):3041–3050).

Thrombomodulin (“TM”) is a transmembrane glycoprotein that hasanticoagulant properties and is predominantly expressed on the lumenalsurface of endothelial cells lining blood vessels (Esmon, N. L. et al.(1982) J. Biol. Chem. 257(2):859–864; Salem, H. H. et al. (1983) J.Biol. Chem. 259(19):12246–12251). The mature, full length TM is a 557amino acid residue modular protein composed of 5 structural domains: anN-terminal, hydrophobic region (residues 1–226); a cysteine-rich region(residues 226–462); a O-glycosylated Ser/Thr-rich region (residues463–497); a hydrophobic transmembrane region (residues 498–521); and aC-terminal cytoplasmic tail (residues 522–557).

The cysteine-rich region includes six repeated structures homologous toepidermal growth factor (“EGF”) precursor, called EGF-like, EGF-homologyor EGF domains. The cysteine-rich region can be further divided into 3domains: the EGF-like repeats 1, 2 and 3 (“EGF123”, residues 226–344),the interdomain loop between EGF3 and EGF4 (residues 345–349), and theEGF-like domains 4, 5 and 6 (“EGF456”, residues 350–462). The functionof EGF456 is to mediate thrombin binding and protein C activation. Onestudy has suggested that the fifth and sixth EGF-like repeats (“EGF5”,residues 390–407, and “EGF6”, residues 427–462, respectively) have thecapacity to bind thrombin (Kurosawa, S. et al. (1988) J. Biol. Chem.263(13):5993–5996); another suggests the EGF456 domain is sufficient toact as cofactor for thrombin-mediated protein C activating activity(Zushi, M. et al. (1989) J. Biol. Chem. 264(18):10351–10353). TheSer/Thr-rich domain enhances EGF456-mediated thrombin binding. The thirdEGF-like repeat (“EGF3”, residues 311–344) is required for theactivation of thrombin-activatable fibrinolysis inhibitor (“TAFI”).Several point mutants in EGF3 have been described that interfere withthe activation of TAFI (Wang, W. et al. (2000) J. Biol. Chem.275(30):22942–22947). The thrombin/TM complex converts protein C toactivated protein C (“APC”), which in turn degrades factors Va andVIIIa, thereby preventing further thrombin generation. Therefore, TMfunctions as a molecular switch converting thrombin from a procoagulantto an anticoagulant.

The K_(m) of protein C for the thrombin/TM complex is reduced 10-foldwhen TM is localized to a membrane surface (Esmon, C. T. (1995) FASEB J.9(10):946–955). The concentration of protein C in blood (0.065 μM) issignificantly below the reported K_(m) (5 μM) for the solubleTM/thrombin complex, therefore establishing that TM on the procoagulantmembrane surface will result in a marked local enhancement of the rateof protein C generation.

TM inhibits thrombosis by a different mechanism from heparin or itsderivatives. Heparin is a cofactor for antithrombin III and inhibitsboth FXa and thrombin through an antithrombin III-dependent mechanism.Thrombus-bound thrombin is protected from the action of antithrombinIII, which limits the antithrombotic efficacy of heparin or lowmolecular weight heparin (“LMWH”) on preexisting clots. This explainsthe failure of heparin or LMWH to inhibit thrombus growth triggered byclot-bound thrombin or prothrombinase in non-human primate studies. Incontrast, recombinant TM attenuates clot induced thrombin generation andfibrin formation in a dose dependent manner (Mohri, M. et al. (1998)Thromb. Haemost. 80(6):925–929). The inhibitory effect of TM isabolished by anti-protein C antibody. Inhibiting clot-bound procoagulantactivity is clinically relevant because clot-bound procoagulant activityresults in more rapid thrombus growth and ultimately in vascularocclusion or thromboembolic complications. Inhibition of thrombus growthallows the endogenous fibrinolytic systems to remove clots more rapidlyand completely. In addition, TM is also expected to be more effectivethan heparin in pathological conditions where plasma antithrombin isdepleted, such as DIC. While both TM and heparin inhibit platelet andfibrinogen consumption in experimental DIC, only TM was effective whenantithrombin III levels were depleted.

SUMMARY OF THE INVENTION

The present invention provides novel fusion proteins, which act asanticoagulants, and comprise a targeting protein, that interacts witheither tissue factor (“TF”) or the factor VIIa/tissue factor(“FVIIa/TF”) complex, which is operably linked to the thrombomodulin(“TM”) EGF456 domain alone or in combination with at least one other TMdomain selected from the group consisting of the N-terminal hydrophobicregion domain, the EGF123 domain, the interdomain loop between EGF3 andEGF4, and the O-glycosylated Ser/Thr-rich domain, or analogs, fragments,derivatives or variants thereof.

The anticoagulant fusion protein of this invention targets and binds TFor the FVIIa/TF complex at the site of injury, localizing TM to theinjury site, and thus preventing thrombus formation and therebyperforming more effectively as an anticoagulant compared to either asoluble anti-TF antibody or soluble TM or fragments of TM. The fusionprotein is more effective than low molecular weight heparin (“LMWH”) inthe treatment of certain diseases including but not limited to sepsis,disseminated intravascular coagulation, ischaemic stroke, deep veinthrombosis, acute coronary syndromes, thrombotic complications followingangioplasty, and coagulopathy in advanced cancer. Further, the fusionprotein has use in microvascular surgery, skin and vein grafts and organtransplants.

In another aspect, the invention provides pharmaceutical compositionsincluding the subject fusion proteins.

In another aspect, the invention provides for a method of protecting apatient against thrombus formation comprising administering atherapeutically effective amount of the fusion protein to said patient,and thereby inhibiting the generation of thrombin without directlyaffecting other coagulation parameters such as the activation andaggregation of platelets.

In another aspect, the invention relates to a method for preventing andtreating deep vein thrombosis (“DVT”) or disseminated intravascularcoagulation (“DIC”) or acute coronary syndrome or cancer with evidenceof coagulopathy in a patient comprising administering a therapeuticallyeffective amount of the fusion protein to said patient.

In another aspect, the invention relates to a method for regulating theinflammatory response in a patient comprising administering atherapeutically effective amount of the fusion protein to said patient.

In yet another aspect, the fusion protein of the invention can be usedto form a non-thrombogenic coating on the surface of medical devicescontacting blood.

In another aspect, the invention relates to a kit comprising a fusionprotein comprising a targeting protein, that binds TF or the FVIIA/TFcomplex, and TM domains. Alternately, the kit may comprise DNA sequencesencoding the fusion protein components.

Also disclosed are methods of making the fusion proteins of theinvention, both recombinant and synthetic.

DESCRIPTION OF THE FIGURES

FIG. 1. Binding of scFv(TF)3e10 to sTF increases the apparent affinityof sTF for FVIIa. The sTF/FVIIa activation assay was performed asdescribed under Example 5 entitled “sTF/FVIIa activation assay” using 2nM FVIIa in the presence and absence of 800 nM scFv(TF)3e10. The sTF wastitrated into the assay and the rate of cleavage of the chromogenicsubstrate (S-2266) was determined. The K_(D) apparent for sTF wascalculated using a standard 4-parameter fit.

FIG. 2. Measurement of binding affinity of scFv(TF)3e10 for sTF. ThesTF/FVIIa assay was as described under Example 5 entitled “sTF/FVIIaactivation assay” using 3 nM sTF and 2 nM FVIIa. The concentration ofsTF used was below the K_(D) for binding to FVIIa. Binding of thescFv(TF)3e10 antibody reduced the K_(D) of sTF for binding to FVIIa,leading to increased formation of the sTF/FVIIa complex and, therefore,the rate of cleavage of the chromogenic substrate S2266. ScFv(TF)3e10was added at increasing concentrations and the increased rate ofreaction was used to determine the K_(D) apparent of the antibody forsTF using a standard 4-parameter fit.

FIG. 3. Microcalorimetry analysis shows scFv(TF)3e10 has a 20-foldhigher affinity for the sTF/FVIIa complex than sTF alone. Isothermaltitration calorimetry was performed using a MicroCal VP-ITC instrument.The sTF/FVIIa complex was preformed by adding a 2.3 fold molar excess ofFVIIai to sTF. Size exclusion chromatography was used to verify that thesTF was completely complexed. For determination of the antibody affinityfor the complex, 1.2 μM sTF/VIIa complex was added to themicrocalorimeter cell and 65 μM scFv(TF)3e10 antibody was added to thesyringe. For determination of the antibody affinity for sTF alone, 10 μMsTF was added to the cell and 141 μM scFv(TF)3e10 was added to thesyringe. Data analysis was done using MicroCal Origin software. The datawas fit to a single binding site.

FIG. 4. scFv(TF)3e10 dose dependently inhibits the FX activation assay.The details of this assay are described under Example 5 entitled “FactorX activation assay”. The IC₅₀ represents the dose required to reach 50%maximum inhibition.

FIG. 5. The fusion protein more potently inhibits coagulation than TFantibody or TMi456 alone. A prothrombin time (PT) assay was performed tocompare the fusion protein with TF antibody or TMi456 alone. Anappropriate volume of concentrated inhibitor, either TF antibody(scFv(TF)3e10), TMi456, or fusion (scFv(TF)3e10-TMi456), was added to100 μl of recombinant human thromboplastin (Ortho Recombiplastin).Approximately 2 minutes later 100 μl reconstituted human plasma wasadded. Coagulation time was determined in a Haemoliance Coagulometer.Dose response curves were generated for each inhibitor and thenregression analysis was used to calculate the concentration (in nM)necessary for a two-fold extension of the clotting time.

FIG. 6. The fusion protein retains full cofactor activity for theactivation of protein C. The assay described under Example 5 entitled“Protein C activation assay (chromogenic)” contained 20 μl TM sample,either TMi456, which contains the EGF domains 4–6 and the interdomainloop between EGF3 and EGF4, or fusion (scFv(TF)3e10-TMi456), 20 μl 1.5μM protein C, and 20 μl 3 nM alpha thrombin. Activation was allowed toproceed for 1 hour. The activation phase was stopped by adding 20 μl0.16 u/ml hirudin. 100 μl of 1 mM S2266 was then added and the A405determined every 10 seconds for 30 minutes. The rate of reaction isdependent on the amount of activated protein C generated. Data isexpressed in mOD/min.

FIG. 7. The rate of protein C activation by the fusion protein isenhanced on TF-containing phospholipid surfaces. The rate of protein Cactivation by TMi456 is not affected by the addition of TF vesicles. Theassay described under Example 5 entitled “Protein C activation assay (onTF-rich surface)” contained 20 μl TM sample, either TMi456 or fusion(scFv(TF)3e10-TMi456), 20 μl of 1.5 μM protein C, 20 μl of 3 nM alphathrombin, and 20 μl of buffer or TF vesicles (Innovin, human recombinantTF, 4×normal concentration for PT). Activation was allowed to proceedfor 1 hour. The activation phase was stopped by adding 20 μl 0.16 u/mlhirudin. 100 μl of 1 mM S2266 was then added and the A405 determinedevery 10 seconds for 30 minutes. The rate of reaction is dependent onthe amount of activated protein C generated. Data is expressed inmOD/min.

FIG. 8. The fusion protein shows greater specificity for TF-inducedcoagulation than TMi456. The activated partial thromboplastin time(APTT) assay is sensitive to inhibitors of the intrinsic and centralpathways of coagulation. Coagulation that occurs in this assay isindependent of TF. The inhibitors, either TF antibody (scFv(TF)3e10),TMi456, or fusion (scFv(TF)3e10-TMi456), were diluted into 50 μlreconstituted human plasma to a final concentration that gave a two-foldextension in the PT assay. The coagulometer then added 50 μl of APTT(Alexin HS) reagent and 50 μl of CaCl₂ reagent (0.02 mol/L) anddetermined the clotting time in seconds.

FIG. 9. The fusion protein more potently inhibits TF-induced whole bloodcoagulation than either of its components alone. Whole blood coagulationwas analyzed using a Haemoscope Thromboelastogragh (TEG) analyzer. Tocitrated whole blood, 120 nM of TF antibody (scFv(TF)3e10), TMi456, orfusion (scFv(TF)3e10-TMi456) was added along with 10 μl of athromboplastin reagent (1:64 dilution) and 20 μl of 0.2M CaCl₂. TheR-value (time to initial fibrin formation) was obtained for each sample.This value was then converted to a % uninhibited control R-value.

FIG. 10. The fusion protein shows a more predictable dose response thanLMWH in a whole blood coagulation assay (TEG). To citrated whole blood,increasing concentrations (15 nM starting and increased by 2×increments)of fusion (scFv(TF)3e10-TMi456), or increasing concentrations (0.15 u/mlstarting and increased by 2×) of enoxaparin (LMWH), were added alongwith 10 μl of a thromboplastin reagent (1:64 dilution) and 20 μl of 0.2MCaCl₂. The R-value (time to initial fibrin formation) was obtained foreach sample and plotted versus relative concentration (set the lowestconcentration as 1 for each (similar R-value), then increase subsequentconcentrations 2×).

FIG. 11. The fusion protein scFV(TF)3e10-TMi456 is efficacious in an invivo model of disseminated intravascular coagulation (“DIC”). TFantibody (scFV(TF)3e10) and fusion (scFV(TF)3e10-TMi456) were evaluatedin the rat thromboembolism model described in Example 8 for (A) percentmortality and (B) morbidity-mortality score. (A) In the vehicle-treatedgroup, the dose of TF used resulted in 60% lethality (LD₆₀).scFv(TF)3e10-TMi456 at 0.7 nmol/kg completely prevented death. Incontrast, scFv(TF)3e10 at 0.7 nmol/kg had no impact on death.scFv(TF)3e10-TMi456 was more efficacious than a 10-fold higher dose ofscFv(TF)3e10. (B) In the vehicle-treated group, the in vivo dose of TFresulted in an average morbidity-mortality score of 2.6, based on thefollowing scoring system: 0=unaffected; 1=mild respiratory distress(recover within 30 min); 2=severe respiratory distress (moribund,recovery required more than 60 min); and 3=death. scFv(TF)3e10-TMi456dose-dependently prevented TF induced death and respiratory distresswith an ED₅₀ value of 0.46 nmol/kg (0.019 mg/kg). scFv(TF)3e10-TMi456 at7.0 nmol/kg completely prevented both death and respiratory distress,and at 0.7 nmol/kg completely prevented death and significantly reducedrespiratory distress. In contrast, scFv(TF)3e10 at 0.7 nmol/kg had noimpact on death and little or no effect on respiratory distress.scFv(TF)3e10-TMi456 was more efficacious than a 10-fold higher dose ofscFv(TF)3e10.

DETAILED DESCRIPTION OF THE INVENTION

The anticoagulant fusion protein of the present invention is comprisedof a targeting protein that interacts with either tissue factor (“TF”)or the factor VIIa/tissue factor (“FVIIa/TF”) complex, which is operablylinked to the thrombomodulin (“TM”) EGF456 domain alone or incombination with at least one other TM domain selected from the groupconsisting of the N-terminal hydrophobic region domain, the EGF123domain, the interdomain loop between EGF3 and EGF4, and theO-glycosylated Ser/Thr-rich domain, or analogs, fragments, derivativesor variants thereof.

Definitions:

In describing the present invention, the following terms are defined asindicated below.

“Recombinant proteins or polypeptides” refer to proteins or polypeptidesproduced by recombinant DNA techniques, i.e., produced from cells,microbial or mammalian, transformed by an exogenous DNA constructencoding the desired polypeptide. Proteins or polypeptides expressed inmost bacterial cultures will be free of glycan. Proteins or polypeptidesexpressed in yeast may have a glycosylation pattern different from thatexpressed in mammalian cells.

“Native” proteins or polypeptides refer to proteins or polypeptidesrecovered from a source occurring in nature. The term “native TM” wouldinclude naturally occurring TM and fragments thereof.

A DNA “coding sequence” is a DNA sequence which is transcribed into mRNAand translated into a polypeptide in a host cell when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ N-terminus anda translation stop codon at the 3′ C-terminus. A coding sequence caninclude prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic DNA, and synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thecoding sequence.

“Fusion protein” is a protein resulting from the expression of at leasttwo operatively linked heterologous coding sequences. The fusion proteinof this invention is comprised of a targeting protein that interactswith either TF or the FVIIa/TF complex, which is operably linked to thethrombomodulin (“TM”) EGF456 domain alone or in combination with atleast one other TM domain selected from the group consisting of theN-terminal hydrophobic region domain, the EGF123 domain, the interdomainloop between EGF3 and EGF4, and the O-glycosylated Ser/Thr-rich domain,or analogs, fragments, derivatives or variants thereof.

“Targeting protein” is a protein that binds to or interacts with anotherprotein or a protein complex. The targeting protein of this invention isa protein that binds to or interacts with TF or the FVIIa/TF complex.For example, an anti-TF or anti-FVIIa/TF complex antibody, is atargeting protein of this invention. Two other examples of targetingproteins are active site inhibited factor VIIa (“FVIIai”), which canbind TF to form an inactive FVIIai/TF complex, and tissue factor pathwayinhibitor (“TFPI”), which can bind to and inactivate the FVIIa/TFcomplex.

“Nucleotide sequence” is a heteropolymer of deoxyribonucleotides (basesadenine, guanine, thymine, or cytosine). DNA sequences encoding thefusion proteins of this invention can be assembled from syntheticcDNA-derived DNA fragments and short oligonucleotide linkers to providea synthetic gene that is capable of being expressed in a recombinantexpression vector. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the nontranscribed strand of cDNA.

“Recombinant expression vector” is a replicable DNA construct usedeither to amplify or to express DNA encoding the fusion proteins of thepresent invention. An expression vector contains DNA control sequencesand a coding sequence. DNA control sequences include promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains and enhancers.Recombinant expression systems as defined herein will express the fusionproteins upon induction of the regulatory elements.

“Transformed host cells” refer to cells that have been transformed andtransfected with exogenous DNA. Exogenous DNA may or may not beintegrated (covalently linked) to chromosomal DNA making up the genomeof the cell. In prokaryotes and yeast, for example, the exogenous DNAmay be maintained on an episomal element, such as a plasmid or stablyintegrated into chromosomal DNA. With respect to eukaryotic cells, astably transformed cell is one in which the exogenous DNA has becomeintegrated into the chromosome replication. This stability isdemonstrated by the ability of the eukaryotic cell lines or clones toproduce a population of daughter cells containing the exogenous DNA

“Thrombomodulin (TM)” refers to an endothelial cell surface glycoproteinthat forms a high affinity complex with thrombin. The genes encodingnative TM (both its genomic form and as cDNA) have been isolated andsequenced from bovine and human (Jackman, R. W. et al. (1986) Proc.Natl. Acad. Sci. USA 83(23):8834–8838 and Jackman, R. W. et al. (1987)Proc. Natl. Acad. Sci. USA 84(18):6425–6429, both of which are hereinincorporated by reference). The sequences for bovine, human and mouse TMexhibit a high degree of homology with one another. The cDNA of human TMencodes a 60.3 kDa protein of 575 amino acids, including a signalsequence of about 18 amino acids, see e.g., U.S. Pat. No. 5,827,824.

When thrombin binds to TM there may be one thousand-fold or moreincrease in the activation rate of protein C which forms theanticoagulant enzyme activated protein C. In addition, when thrombin isbound to TM, thrombin no longer works as a procoagulant enzyme.Specifically, thrombin-catalyzed fibrin formation, factor V activation,and platelet activation, are all inhibited in the presence of TM. Thus,TM converts thrombin into a physiological anticoagulant.

“Thrombomodulin (TM) domain” refers to a discrete amino acid sequencethat can be associated with a particular function or characteristic ofTM, such as a characteristic tertiary structural unit. The full-lengthTM gene (SEQ ID NO; 4) encodes a precursor or pro-polypeptide containingthe following domains: amino acids 18–1 is the signal sequence; aminoacids 1–226 is the N-terminal hydrophobic region; amino acids 227–462 isthe cysteine-rich region; consisting of 6 tandem EGF-like repeats joinedby small interdomain peptides or loops; amino acids 463–497 is anO-glycosylated Ser/Thr-rich region; amino acids 498–521 is a hydrophobictransmembrane region; and amino acids 522–557 is the C-terminalcytoplasmic tail. The cysteine-rich region can be further divided into 3domains: amino acids 226–344 is EGF123, consisting of the EGF-likerepeats 1, 2 and 3 (residues 226–344); amino acids 345–349 is theinterdomain loop between EGF3 and EGF4; and amino acids 350–462 isEGF456, consisting of the EGF-like domains 4, 5 and 6. See e.g., Yost,C. S. et al. (1983) Cell 34(3):759–766; Wen, D. Z. et al. (1987)Biochemistry 26(14):4350–4357; and Wang, W. et al. (2000), supra, all ofwhich are incorporated herein by reference. The amino acid sequence ofnative thrombomodulin is given in SEQ ID NO: 5.

The terms “analog”, “fragment”, “derivative”, and “variant”, whenreferring to the fusion proteins of this invention, as well as thetargeting proteins and the TM domain(s), means analogs, fragments,derivatives, and variants of the fusion proteins, targeting proteins andTM domain(s) which retain substantially the same biological function oractivity, as described further below.

An “analog” includes a pro-polypeptide which includes within it, theamino acid sequence of the fusion protein of this invention. The activefusion protein of this invention can be cleaved from the additionalamino acids that complete the pro-fusion protein molecule by natural, invivo processes or by procedures well known in the art such as byenzymatic or chemical cleavage. For example, native TM is naturallyexpressed as a 575 amino acid pro-polypeptide which is then processed invivo to release the 557 amino acid active mature polypeptide.

A “fragment” is a portion of the fusion protein, targeting protein or TMdomain(s) which retains substantially similar functional activity, asshown in the in vitro assays disclosed herein as described furtherbelow.

A “derivative” includes all modifications to the fusion protein whichsubstantially preserve the functions disclosed herein and includeadditional structure and attendant function, e.g., PEGylated fusionproteins which have greater half-life, O-glycosylated fusion proteinsmodified by the addition of chondroitin sulfate, and biotinylated fusionproteins, as described further below.

“Substantially similar functional activity” and “substantially the samebiological function or activity” each means that the degree ofbiological activity that is within about 30% to 100% or more of thatbiological activity demonstrated by the polypeptide to which it is beingcompared when the biological activity of each polypeptide is determinedby the same procedure or assay. For example, a fusion protein or TMdomain(s) that has substantially similar functional activity to thefusion protein of Example 2 (SEQ ID NO:2) is one that, when tested inthe protein C activation assay (chromogenic) described in Example 5,demonstrates accumulation of activated protein C. A targeting proteinthat has substantially similar functional activity to the anti-TFantibody of Example 1 (SEQ ID NO:1) is one that, when tested in thesTF/FVIIa assay or FX activation assays described in Example 5,demonstrates the ability to bind to or neutralize TF or the FVIIa/TFcomplex.

“Similarity” between two polypeptides is determined by comparing theamino acid sequence and its conserved amino acid substitutes of onepolypeptide to the sequence of a second polypeptide. Such conservativesubstitutions include those described above in The Atlas of ProteinSequence and Structure 5 by Dayhoff (1978) and by Argos (1989) EMBO J.8:779–785. For example, amino acids belonging to one of the followinggroups represent conservative changes:

Ala, Pro, Gly, Gln, Asn, Ser, Thr: Cys, Ser, Tyr, Thr; Val, Ile, Leu,Met, Ala, Phe; Lys, Arg, His; Phe, Tyr, Trp, His; and Asp, Glu.

All other technical terms used herein have the same meaning as iscommonly used by those skilled in the art to which the present inventionbelongs.

Targeting Protein:

The targeting protein of this invention is a protein that has theability to specifically bind to a particular preselected targetmolecule, e.g., TF or the FVIIa/TF complex, and then serves to directthe fusion protein to a cell or tissue bearing the preselected targetmolecule.

In one embodiment of this invention, the targeting protein is anantibody that can bind to and neutralize TF or the FVIIa/TF complex.“Antibody” as used herein includes intact immunoglobulin (“Ig”)molecules, as well as fragments thereof, such as Fab, F(ab′)₂, and Fv,which are capable of binding an epitope of TF or the FVIIa/TF complex.Typically, at least 6, 8, 10, or 12 contiguous amino acids are requiredto form an epitope. However, epitopes which involve non-contiguous aminoacids may required more, e.g. at least 15, 25, or 50 amino acids.

Typically, an antibody that binds specifically to TF or the FVIIa/TFcomplex provides a detection signal at least 5-, 10-, or 20-fold higherthan a detection signal provided with other proteins when used in animmunochemical assay. Preferably, antibodies that bind specifically toTF or the FVIIa/TF complex do not detect other proteins inimmunochemical assays and can immunoprecipitate TF or the FVIIa/TFcomplex from solution.

TF or the FVIIa/TF complex can be used to immunize a mammal, such as amouse, rat, rabbit, guinea pig, monkey, or human to produce polyclonalantibodies. If desired, TF or the FVIIa/TF complex can be conjugated toa carrier protein, such as bovine serum albumin, thyroglobulin, andkeyhole limpet hemocyanin. Depending on the host species, variousadjuvants can be used to increase the immunological response. Suchadjuvants include, but are not limited to, Freund's adjuvant, mineralgels (e.g., aluminum hydroxide), and surface active substances (e.g.,lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Cornybacterium parvum areespecially useful.

Monoclonal antibodies that bind specifically to TF or the FVIIa/TFcomplex can be prepared using any technique which provides for theproduction of antibody molecules by continuous cell lines in culture.These techniques include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler et al. (1985) Nature 256:495–497; Kozbor et al. (1985)J. Immunol. Methods 81:31–42; Cote et al. (1983) Proc. Natl. Acad. Sci.USA 80:2026–2030; and Cote et al. (1984) Mol. Cell Biol. 62:109–120).

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity, can be used (Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81:6851–6855; Neuberger et al. (1984) Nature 312:604–608;Takeda et al. (1985) Nature 314:452–454). Monoclonal and otherantibodies also can be “humanized” to prevent a patient from mounting animmune response against the antibody when it is used therapeutically.Such antibodies may be sufficiently similar in sequence to humanantibodies to be used directly in the fusion protein or may requirealteration of a few key residues. Sequence differences between rodentantibodies and human sequences can be minimized by replacing residueswhich differ from those in the human sequences by site directedmutagenesis of individual residues or by grafting of entirecomplementarity determining regions. Alternatively, humanized antibodiescan be produced using recombinant methods, as described in GB2188638B.Antibodies that bind specifically to TF or the FVIIa/TF complex cancontain antigen-binding sites which are either partially or fullyhumanized, as disclosed in U.S. Pat. No. 5,565,332.

Alternatively, techniques described for the production of single chainantibodies can be adapted using methods known in the art to producesingle chain antibodies that specifically bind to TF or the FVIIa/TFcomplex. Antibodies with related specificity, but of distinct idiotypiccomposition, can be generated by chain shuffling from randomcombinatorial Ig libraries (Burton (1991) Proc. Nat. Acad. Sci. USA88:11120–11123).

Single chain antibodies also can be constructed using a DNAamplification method, such as PCR, using hybridoma cDNA as a template(Thirion et al. (1996) Eur. J. Cancer Prev. 5:507–511). Single chainantibodies can be mono- or bispecific, and can be bivalent ortetravalent. Construction of tetravalent, bispecific single chainantibodies is taught, for example, in Coloma and Morrison (1997) Nat.Biotechnol. 15:159–163. Construction of bivalent, bispecific singlechain antibodies is taught in Mallendar and Voss (1994) J. Biol. Chem.269:199–216.

A nucleotide sequence encoding a single chain antibody can beconstructed using manual or automated nucleotide synthesis, cloned intoan expression construct using standard recombinant DNA methods, andintroduced into a cell to express the coding sequence. Alternatively,single chain antibodies can be produced directly using, for example,filamentous phage display technology (Verhaar et al. (1995) Int. J.Cancer 61:497–501; and Nicholls et al. (1993) J. Immunol. Meth.165:81–91).

Antibodies that bind specifically to TF or the FVIIa/TF complex can alsobe produced by inducing in vivo production in the lymphocyte populationor by screening Ig libraries or panels of highly specific bindingreagents as disclosed in the literature. (Orlandi et al. (1989) Proc.Natl. Acad. Sci. USA 86:3833–3837; Winter et al. (1991) Nature349:293–299).

In another embodiment of this invention, the targeting protein is atargeting moiety other than an antibody that can bind to and neutralizeTF. Two such examples are active site inhibited factor FVIIa (FVIIai)and tissue factor pathway inhibitor (TFPI).

Both FVIIa and FVIIai form a high affinity complex with TF (Sorenson, B.B. and Rao, L. V. (1998) Blood Coagul. Fibrinolysis 9(Suppl 1):S67–71).FVIIai is a TF neutralizing anticoagulant that acts by competing withendogenous FVIIa for binding to exposed TF. FVIIai inhibits the abilityof proteolytically active FVIIa to form a competent FVIIa-TF complex andin this way inhibits initiation of coagulation. By genetically fusing TMdomains to FVIIai, TM could be targeted to TF-rich prothromboticsurfaces.

The cDNA encoding human FVII has been isolated and sequenced (Hagen, H.S. et al. (1986) Proc. Natl. Acad. Sci. USA 83(8):2412–2416, which isincorporated herein by reference). The human FVII cDNA can be made bystandard recombinant DNA techniques starting from mRNA isolated fromhuman liver. FVIIai can be made by mutating the active site serine bystandard recombinant DNA techniques or by chemically treatingcatalytically active FVIIa with a peptidyl chloromethylketone, whichirreversibly modifies and inhibits the active site.

TFPI targets and inhibits the FVIIa/TF complex in a FXa dependentfashion (Salemink, I. et al. (1999) J. Biol. Chem. 274(40):28225–28232).TFPI first binds to FXa and then the TFPI-FXa complex binds to andinhibits the FVIIa/TF complex. By genetically fusing TM domains to TFPI,TM could be targeted to TF-rich prothrombotic surfaces.

The cDNA encoding human TFPI has been isolated and sequenced (Wun, T. C.et al. (1988) J. Biol. Chem. 263(13):6001–6004, which is incorporatedherein by reference). The human TFPI cDNA can be made by standardrecombinant DNA techniques starting from mRNA isolated from human liver.

The targeting protein of this invention (i.e, antibodies or otherrelevant proteins) can be expressed and purified by methods well knownin the art. For example, antibodies and proteins can be affinitypurified by passage over a column to which TF is bound. The boundantibodies or proteins can then be eluted from the column using a bufferwith a high salt concentration.

In one preferred embodiment of this invention, the targeting protein isa TF-binding scFv antibody that inhibits activation of FX by theFVIIa/TF complex and does not compete with FVIIa binding. In order toproduce the TF-binding scFv antibody, the human antibody libraryHuPhaBL3, which was displayed on filamentous phage, was selected againstimmobilized soluble TF. Antibodies from TF binding phage wereoverexpressed in E. coli and affinity purified using an e-tag column.The purified antibodies were further characterized using BIAcore, a sTFdependent factor VIIa assay (sTF/FVIIa assay), a FX activation assay,and the PT assay. The sequence of the TF-binding scFv antibody,designated scFv(TF)3e10, is shown in Example 1 and corresponds to SEQ IDNO:1. The isolation, production and characterization of the TF-bindingscFV antibody are described in greater detail below.

Thrombomodulin:

The TM domain(s) portion of the fusion protein acts as a cofactor forthrombin catalyzed activation of protein C, which in turn degradesfactors Va and VIIIa thereby preventing further thrombus formation. Thedomains of TM include e.g., the N-terminal hydrophobic region domain,the EGF123 domain, the interdomain loop between EGF3 and EGF4, theEGF456 domain, and the O-glycosylated Ser/Thr-rich region domain. TheEGF456 domain, in particular, mediates thrombin binding and protein Cactivation (Kurosawa, S. et al. (1988), supra; and Zushi, M. et al.(1989) supra). In preferred embodiments of this invention, the TMdomain(s) portion of the fusion protein comprises the EGF456 domainalone or in combination with one or more of the other TM domains. Instill more preferred embodiments of this invention, the EGF456 domaincontains point mutations that render the protein more resistant tooxidative damage and proteases and/or increase its catalytic efficiency.

The full length DNA sequence encoding human TM facilitates thepreparation of genes and is used as a starting point to construct DNAsequences encoding TM peptides and fusion proteins containing TM andfragments/peptides of TM.

The full-length gene for TM can be prepared by several methods. Humangenomic libraries are commercially available. Oligonucleotide probes,specific to these genes, can be synthesized using the published genesequence. Methods for screening genomic libraries with oligonucleotideprobes are known. The publication of the gene sequence for TMdemonstrates that there are no introns within the coding region. Thus, agenomic clone provides the necessary starting material to construct anexpression plasmid for TM using known methods.

A TM encoding DNA fragment can be retrieved by taking advantage ofrestriction endonuclease sites that have been identified in regionswhich flank or are internal to the gene. (Jackman, R. W. et al. (1987),supra). Alternately, the full-length genes can also be obtained from acDNA bank. For example, messenger RNA prepared from endothelial cellsprovides suitable starting material from the preparation of cDNA.Methods for making cDNA banks are well known (see e.g., Sambrook, J. F.et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory (1989), which is herein incorporated by reference).

Fusion Protein:

The anticoagulant fusion protein of this invention comprises a targetingprotein that binds to either TF or the FVIIa/TF complex, and which isoperably linked to the TM EGF456 domain alone or in combination with atleast one other TM domain selected from the group consisting of theN-terminal hydrophobic region domain, the EGF123 domain, the interdomainloop between EGF3 and EGF4, and the O-glycosylated Ser/Thr-rich domain,or analogs, fragments, derivatives or variants thereof. The fusionprotein can comprise the targeting protein linked with the domains of TMin any combination.

In one particularly preferred embodiment, the fusion protein comprisesan antibody that binds TF, operably linked to the TM EGF456 domain andthe interdomain loop between EGF3 and EGF4 (“TMi456”), or analogs,fragments, derivatives or variants thereof.

The fusion protein of the present invention includes, but it not limitedto, constructs in which the C-terminal portion of a single chainantibody is fused to the N-terminal portion of an analog, fragment,derivative or variant of a TM domain(s), the C-terminal portion of anIgG antibody is fused to the N-terminal portion of an analog, fragment,derivative or variant of a TM domain(s), the C-terminal portion of anFab antibody is fused to the N-terminal portion of an analog, fragment,derivative or variant of a TM domain(s), the N-terminal portion of asingle chain antibody is fused to the C-terminal portion of an analog,fragment, derivative or variant of a TM domain(s), the N-terminalportion of an IgG antibody is fused to the C-terminal portion of ananalog, fragment, derivative or variant of a TM domain(s), theN-terminal portion of an Fab antibody is fused to the C-terminal portionof an analog, fragment, derivative or variant of a TM domain(s), morethan one single chain antibody is fused to both the N-terminal and theC-terminal portions of an analog, fragment, derivative or variant of aTM domain(s), more than one IgG antibody is fused to both the N-terminaland the C-terminal portions of an analog, fragment, derivative orvariant of a TM domain(s), more than one Fab antibody is fused to boththe N-terminal and the C-terminal portions of an analog, fragment,derivative or variant of a TM domain(s), more than one analog, fragment,derivative or variant of a TM domain(s) is fused to both the N-terminaland the C-terminal portions of a single chain antibody, more than oneanalog, fragment, derivative or variant of a TM domain(s) is fused toboth the N-terminal and the C-terminal portions of an IgG antibody, morethan one analog, fragment, derivative or variant of a TM domain(s) isfused to both the N-terminal and the C-terminal portions of an Fabantibody, one or more than one analog, fragment, derivative or variantof a TM domain(s) is fused to both the N-terminal and the C-terminalportions of a dimeric single chain antibody.

The fusion proteins of the present invention include the fusion proteinsof Examples 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3), as well as those fusionproteins having insubstantial variations in sequence from them. An“insubstantial variation” would include any sequence, substitution, ordeletion variant that maintains substantially at least one biologicalfunction of the polypeptides of this invention, preferably cofactoractivity for thrombin-mediated protein C activation. These functionalequivalents may preferably include fusion proteins which have at leastabout a 90% identity to the fusion proteins of SEQ ID NOs:2 or 3, andmore preferably at least a 95% identity to the fusion proteins of SEQ IDNOs:2 or 3, and still more preferably at least a 97% identity to thefusion proteins of SEQ ID NOs:2 or 3, and also include portions of suchfusion proteins having substantially the same biological activity.However, any fusion protein having insubstantial variation in amino acidsequence from the fusion proteins of SEQ ID NOs:2 and 3 thatdemonstrates functional equivalency as described further herein isincluded in the description of the present invention.

In another embodiment, the fusion protein comprises an antibody thatbinds TF operably linked to TM domain EGF3, which is required toactivate thrombin-activatable fibrinolysis activator (TAFI).

Analogs, Fragments, Derivatives and Variants:

An analog, fragment, derivative, or variant of the fusion proteins, aswell as targeting proteins or TM domain(s), of the present invention maybe: (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code; or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature fusion protein isfused with another compound, such as a compound to increase thehalf-life of the fusion protein (for example, polyethylene glycol), or(iv) one in which additional amino acids are fused to the mature fusionprotein, such as a leader or secretory sequence or a sequence which isemployed for purification of the mature fusion protein, or (v) one inwhich the polypeptide sequence is fused with a larger polypeptide, i.e.,human albumin, an antibody or Fc, for increased duration of effect. Suchanalogs, fragments, derivatives, and variants are deemed to be withinthe scope of those skilled in the art from the teachings herein.

Preferably, the derivatives of the present invention will containconservative amino acid substitutions (defined further below) made atone or more predicted, preferably nonessential amino acid residues. A“nonessential” amino acid residue is a residue that can be altered fromthe wild-type sequence of a protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Non-conservative substitutions would not be made forconserved amino acid residues or for amino acid residues residing withina conserved protein domain, unless the non-conservative substitutionsare made to render the resulting fusion protein more resistant tooxidative damage and proteases and/or increase its catalytic efficiency.Fragments or biologically active portions include polypeptide fragmentssuitable for use as a medicament, as a research reagent, and the like.Fragments include peptides comprising amino acid sequences sufficientlysimilar to or derived from the amino acid sequences of a fusion proteinof this invention and exhibiting at least one activity of thatpolypeptide, but which include fewer amino acids than the full-lengthpolypeptides disclosed herein. Typically, biologically active portionscomprise a domain or motif with at least one activity of thepolypeptide. A biologically active portion of a polypeptide can be apeptide that is, for example, 5 or more amino acids in length. Suchbiologically active portions can be prepared synthetically or byrecombinant techniques and can be evaluated for one or more of thefunctional activities of a polypeptide of this invention by meansdisclosed herein and/or well known in the art.

Moreover, preferred derivatives of the present invention include maturefusion proteins that have been fused with another compound, such as acompound to increase the half-life of the polypeptide and/or to reducepotential immunogenicity of the polypeptide (for example, polyethyleneglycol, “PEG”). The PEG can be used to impart water solubility, size,slow rate of kidney clearance, and reduced immunogenicity to the fusionprotein. See e.g., U.S. Pat. No. 6,214,966. In the case of PEGylations,the fusion of the fusion protein to PEG can be accomplished by any meansknown to one skilled in the art. For example, PEGylation can beaccomplished by first introducing a cysteine mutation into the fusionprotein, followed by site-specific derivatization with PEG-maleimide.The cysteine can be added to the C-terminus of the peptides. See, e.g.,Tsutsumi et al. (2000) Proc. Natl. Acad. Sci. USA 97(15):8548–8553.Another modification which can be made to the fusion protein involvesbiotinylation. In certain instances, it may be useful to have the fusionprotein biotinylated so that it can readily react with streptavidin.Methods for biotinylation of proteins are well known in the art.Additionally, chondroitin sulfate can be linked with the fusion protein.

Variants of the fusion proteins, targeting proteins and TM domain(s) ofthis invention include polypeptides having an amino acid sequencesufficiently similar to the amino acid sequence of the original fusionproteins, targeting proteins and TM domain(s). The term “sufficientlysimilar”means a first amino acid sequence that contains a sufficient orminimum number of identical or equivalent amino acid residues relativeto a second amino acid sequence such that the first and second aminoacid sequences have a common structural domain and/or common functionalactivity. For example, amino acid sequences that contain a commonstructural domain that is at least about 45%, preferably about 75%through 98%, identical are defined herein as sufficiently similar.Preferably, variants will be sufficiently similar to the amino acidsequence of the preferred fusion proteins of this invention. Variantsinclude variants of fusion proteins encoded by a polynucleotide thathybridizes to a polynucleotide of this invention or a complement thereofunder stringent conditions. Such variants generally retain thefunctional activity of the fusion proteins of this invention. Librariesof fragments of the polynucleotides can be used to generate a variegatedpopulation of fragments for screening and subsequent selection. Forexample, a library of fragments can be generated by treating adouble-stranded PCR fragment of a polynucleotide with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double-stranded DNA, renaturing the DNA to formdouble-stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single-stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method,one can derive an expression library that encodes N-terminal andinternal fragments of various sizes of the fusion proteins of thisinvention.

Variants include fusion proteins, as well as targeting proteins and TMdomain(s), that differ in amino acid sequence due to mutagenesis.Variants that have cofactor activity for thrombin-mediated protein Cactivation can be identified by screening combinatorial libraries ofmutants, for example truncation or point mutants, of the fusion proteinsor TM domain(s) of this invention using the protein C activation assaydescribed in Example 5. Variants that have TF- or FVIIa/TFcomplex-binding activity can be identified by screening combinatoriallibraries of mutants, for example truncation or point mutants, of thefusion proteins or targeting proteins of this invention using thesTF/FVIIa assay or FX activation assays of Example 5 described inExample 5. In addition, bioequivalent analogs of the fusion proteins mayalso be constructed by making various substitutions on residues orsequences in the TM domain(s) portion of the fusion protein which canrender the fusion protein more oxidation damage or protease resistant,see e.g., U.S. Pat. No. 5,827,824, or increase the catalytic efficiencyof the fusion protein, see e.g., Adler, M. et al. (1995) J. Biol. Chem.270(40):23366–23372, and PCT patent application WO01/98352, publishedDec. 27, 2001, all of which are fully incorporated herein by reference.

In one embodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential variant amino acid sequences is expressible as individualpolypeptides, or, alternately, as a set of larger fusion proteins (forexample, for phage display) containing the set of sequences therein.There are a variety of methods that can be used to produce libraries ofpotential variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential variant sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984a) Annu. Rev. Biochem. 53:323;Itakura et al. (1984b) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of fusion proteins, as wellas targeting proteins and TM domain(s), for cofactor activity forthrombin-mediated protein C activation or TF- or FVIIa/TFcomplex-binding activity. The most widely used techniques, which areamenable to high throughput analysis for screening large gene librariestypically include cloning the gene library into replicable expressionvectors, transforming appropriate cells with the resulting library ofvectors and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique that enhances the frequency of functionalmutants in the libraries, can be used in combination with the screeningassays to identify the desired variants.

Producing Fusion Proteins:

The fusion protein of this invention is produced by fusing the targetingprotein to, or otherwise binding it to, the TM domain(s) or analogs,fragments, derivatives or variants thereof by any method known to thoseskilled in the art. The two components may be chemically bonded togetherby any of a variety of well-known chemical procedures. For example, thelinkage may be by way of heterobifunctional cross-linkers, e.g., SPDP,carbodiimide, glutaraldehyde, or the like.

In a more preferred embodiment, the targeting protein of this inventioncan be fused to the TM domain(s) by recombinant means such as throughthe use of recombinant DNA techniques to produce a nucleic acid whichencodes both the targeting protein and the polypeptide encoding the TMdomain(s) and expressing the DNA sequence in a host cell such as E. colior a mammalian cell. The DNA encoding the fusion protein may be clonedin cDNA or in genomic form by any cloning procedure known to thoseskilled in the art. See for example, Sambrook, J. F. et al. (1989)supra.

In the case where the targeting protein is an antibody, once a DNAsequence has been identified that encodes a Fv region which whenexpressed shows specific binding activity, fusion proteins comprisingthat Fv region may be prepared by methods known to one of skill in theart. Thus, for example, Chaudhary, V. K. et al. (1989) Nature 339(6223):394–397; Batra, J. K. et al. (1990) J. Biol. Chem. 265(25):15198–15202;Batra, J. K. et al. (1989) Proc. Natl. Acad. Sci. USA 86(21):8545–8549;Chaudhary, V. K. et al. (1990) Proc. Natl. Acad. Sci. USA87(3):1066–1070, all incorporated by reference, describe the preparationof various single chain antibody fusion proteins. The Fv region may befused directly to the TM domain(s) or may be joined via a linkersequence. The linker sequence may be present simply to provide spacebetween the targeting moiety and the TM domain(s) or to facilitatemobility between these regions to enable them to each attain theiroptimum conformation. The DNA sequence comprising the connector may alsoprovide sequences (such as primer or restriction sites) to facilitatecloning or may preserve the reading frame between the sequence encodingthe targeting moiety and the sequence encoding the TM domain(s). Thedesign of such connector peptides will be well known to those of skillin the art.

In the present invention, linker sequences can be used for linking thetargeting protein with the TM domain(s). In one preferred embodiment ofthe present invention, two linker sequences are used in constructing afusion protein comprised of a single chain antibody and the TM EGF456domain and the interdomain loop between EGF3 and EGF4 (TMi456). Thefirst links the heavy and light domains of the single chain antibody.The first linker sequence is 5 amino acids in length. It will beapparent that other short linker sequences, from 0 to 10 amino acids maybe used. The second linker in the present invention is a 15 amino acidlinker that links the antibody to the TM domain(s). It will be apparentto those of skill in the art that many different linker sequences may beused and still result in a fusion protein which retains anticoagulantactivity and the activation of protein C. Modifications of the existinglinker will be aimed at maximizing the enhancement of protein Cactivation on TF-containing phospholipid surfaces.

In a preferred approach, the single chain antibody was prepared using aphage display library. In the first step of constructing a phage displaylibrary, the variable genes (V_(H) (from IgM) V_(κ) and V_(L)) were PCRcloned from pooled mRNA from human bone marrow, lymph node and spleenusing a set of family specific primers. The resultant pCITE-V_(H)(3.8×10⁹ members), pZ604-V_(κ) (1.6×10⁷) and pZ604-V_(L) (3.2×10⁷)libraries represent a permanent and high diversity of V genes. The V_(H)genes were amplified from pCITE-V_(H) library. The V_(κ) and V_(L) geneswere PCR amplified from the pZ604-V_(κ) and pZ604-V_(L) library withreverse J_(H) and linker sequence at the 5′end. The gel purified V_(H),V_(κ) and V_(L) containing PCR products were then spliced together tomake the scFv gene repertoire. The scFV gene repertoire was cloned to aphagemid vector pZ603, and the ligation product was electroporated intocompetent TG1 E. coli cells to generate the scFV phage display library,HuPhabL3, with 5.2×10⁹ individual transformants (Kay, B. K. et al.(1996) Phage Display of Peptides and Proteins: A Laboratory Manual,Academic Press, San Diego Calif.; Marks, J. D. et al. (1991) J. Mol.Biol. 222(3):581–597; Sheets, M. D. et al. (1998) Proc. Natl. Acad. Sci.USA 95(11):6157–6162).

In a preferred embodiment of the present invention, a single chainantibody (scFv(TF)3e10) was prepared which has a single V_(H)/V_(L)binding site for TF. The amino acid sequence of scFv(TF)3e10 (SEQ IDNO:1), is depicted in Example 1.

In a preferred embodiment of the present invention, a PCR fragmentcontaining the TMi456 sequence (with M388L and H381G mutations) flankedby NotI sites was subcloned into the NotI site of pZ612/3e10 (abacterial expression vector for scFv(TF)3e10 based on pCANTAB5 fromPharmacia). Herein, point mutations in the TM portions of the fusionproteins of the invention are specified the single letter designation ofthe amino acid residue of native TM, followed by the amino acid positionnumber in mature TM and the single letter designation of the amino acidmutation. For example, M388L indicates that the methionine at amino acidposition 388 of mature TM has been changed to leucine. The NotI site isbetween the antibody sequence and the e-tag sequence. This generated abacterial expression construct (pKM101) for a fusion protein comprisedof the scFV(TF)3e10—a 15 amino acid linker—TMi456, followed by the e-tagsequence. To generate a mammalian expression vector a PCR fragment wasfirst generated from the pKM101 template. This fragment was designed forligation into the StuI/MscI sites of the TM expression vector pTHR525.This generated a vector (pKM113) that had the Solulin signal sequencefollowed by the sequence for the mature fusion protein followed by thee-tag sequence. The vector contains the ampicillin resistance gene andthe hydromycin and DHFR selection markers. The expression is driven bythe MPSV LTR promoter. Site directed mutagenesis was performed on thisvector to include the R456G and H457Q mutations which confer proteaseresistance to the TM portion. The resulting vector is referred to aspKM115. The pMK115 vector had a 15 amino acid linker separating theV_(H) and V_(L) domains and another 15 amino acid linker separating theV_(L) domain from the TMi456. The linker separating the V_(H) and V_(L)was decreased to 5 amino acids to drive the formation of a higheravidity dimer, referred to as pHM115.5. The fusion protein encoded bypHM115.5, scFv(TF)3e10-TMi456 (SEQ ID NO: 2), is depicted in Example 2.An additional vector, pKM125, was generated using standard recombinantDNA technology by deleting 3 amino acids (GAP) between the 5 amino acidlinker separating the V_(H) and V_(L) domains and deleting the e-tag atthe C-terminus of the fusion protein. The resulting fusion protein,scFv(TF)3e10-TMi456Δ (SEQ ID NO:3), is depicted in Example 3.

Expression and Purification of Fusion Proteins:

There are several ways to express the recombinant fusion proteins invitro, including E. coli, baculovirus, yeast mammalian cells or otherexpression systems. Methods for the expression of cloned genes inbacteria are well known. To obtain high level expression of a clonedgene in a prokaryotic system, it is essential to construct expressionvectors which contain, at the minimum, a strong promoter to direct mRNAtranscription termination. Examples of regulatory regions suitable forthis purpose are the promoter and operator region of the E. colibeta-glucosidase gene, the E. coli tryptophan biosynthetic pathway, orthe leftward promoter from phage Lambda. The inclusion of selectionmarkers in DNA vectors transformed in E. coli is useful. Examples ofsuch markers include the genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

Of the higher eukaryotic cell systems useful for expression of thefusion proteins and analogs, thereof, there are numerous cell systems toselect from. Illustrative examples of mammalian cell lines include butare not limited to RPMI 7932, VERO and HeLa cells, Chinese hamster ovary(CHO) cell lines, WI38, BHK, COS-7, C127 or MDCK cell lines. A preferredmammalian cell lines is CHL-1. When CHL-1 is used hygromycin is includedas a eukaryotic selection marker. CHL-1 cells are derived from RPMI 7032melanoma cells, a readily available human cell line. The CHL-1 cell linehas been deposited with ATCC according to conditions of the BudapestTreaty and has been assigned #CRL 9446, deposited Jun. 18, 1987. Cellssuitable for use in this invention car commercially available from theATCC. Illustrative cell lines include Spodoptera frugiperda and Bombyxmori.

The prokaryotic system, E. coli, is not able to do post-translationalmodification, such as glycosylation. In addition proteins with complexdisulfide patterns are often misfolded when expressed in E. coli. Forthe fusion protein described herein there was a marked reduction in thethrombomodulin cofactor activity when expressed in E. coli although bothactivities were still present. With the prokaryotic system, theexpressed protein is either present in the cell cytoplasm in aninsoluble form so-called inclusion bodies, found in the soluble fractionafter the cell has lysed, or is directed into the periplasm by additionof appropriate secretion signal sequences. If the expressed protein isin insoluble inclusion bodies, solubilization and subsequent refoldingof the inclusion bodies is usually required.

Many prokaryotic expression vectors are known to those of skill in theart such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden),pKK233-2 (Clontech, Palo Alto, Calif., USA), and pGEM1 (Promega Biotech,Madison, Wis., USA), which are commercially available.

Promoters commonly used in recombinant microbial expression systemsinclude the beta-lactamase (penicillinase) and lactose promoter system(Chang, A. C. et al. (1978) Nature 275(5681):617–624; Goeddel, D. V. etal. (1979) Nature 281 (5732):544–548), the tryptophan (trp) promotersystem (Goeddel, D. V. et al. (1980) Nucl. Acids Res. 8(18):4057–4074)and tac promoter (Maniatis, T. et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory (1982)). Another useful bacterialexpression system employs the lambda phage pL promoter and clts857thermoinducible repressor (Bernard, H. U. et al. (1979) Gene 5(1):59–76;Love, C. A. et al. (1996) Gene 176(1–2):49–53). Recombinant fusionproteins may also be expressed in yeast hosts such as Saccharomycescerevisiae. It usually gives the ability to do variouspost-translational modifications. The expressed fusion protein can besecreted into the culture supernatant where not many other proteinsreside, making purification easier. Yeast vectors for expression of thefusion proteins in this invention contain certain requisite features.The elements of the vector are generally derived from yeast and bacteriato permit propagation of the plasmid in both. The bacterial elementsinclude an origin of replication and a selectable marker. The yeastelements include an origin of replication sequence (ARS), a selectablemarker, a promoter, and a transcriptional terminator.

Suitable promoters in yeast vectors for expression include the promotersof TRP1 gene, the ADH1 or ADHII gene, acid phosphatase (PH03 or PH05)gene, isocytochrome gene, or the promoters involved with the glycolyticpathway, such as the promoter of enolase, glyceraldehyde-3-phosphatedehydrogenase (GADPH), 3-phosphoglycerate kinase (PGK), hexokinase,pyruvate kinase, triosephosphate isomerase and phosphoglucose isomerase(Hitzeman, R. A. et al. (1980) J. Biol. Chem. 255(24):12073–12080; Hess,B. et al. (1968) J. Adv. Enzyme Reg. 7:149–167; and Holland, M. J. andHolland, J. P. (1978) Biochemistry 17(23):4900–4907).

Commercially available yeast vectors include pYES2, pPIC9 (Invitrogen,San Diego, Calif.), Yepc-pADH2a, pYcDE-1 (Washington Research, Seattle,Wash.), pBC102-K22 (ATCC #67255), and YpGX265GAL4 (ATCC #67233).Mammalian cell lines including but not limited to COS-7, L cells, C127,3T3, Chinese Hamster Ovary (CHO), HeLa, BHK, CHL-1, NSO, and HEK293 canbe employed to express the recombinant fusion proteins in thisinvention. The recombinant proteins produced in mammalian cells arenormally soluble and glycosylated and have authentic N-termini.Mammalian expression vectors may contain non-transcribed elements suchas an origin of replication, promoter and enhancer, and 5′ or 3′nontranslated sequences such as ribosome binding sites, apolyadenylation site, acceptor site and splice donor, andtranscriptional termination sequences. Promoters for use in mammalianexpression vectors usually are for example viral promoters such asPolyoma, Adenovirus, HTLV, Simian Virus 40 (SV 40), and humancytomegalovirus (CMV).

Depending on the expression system and host selected, a homogeneousrecombinant fusion protein can be obtained by using various combinationsof conventional chromatography used for protein purification. Theseinclude: immunoaffinity chromatography, reverse phase chromatography,cation exchange chromatography, anion exchange chromatography,hydrophobic interaction chromatography, gel filtration chromatography,and HPLC If the expression system secretes the fusion protein into thegrowth media, the protein can be purified directly from the media. Ifthe fusion protein is not secreted, it is isolated from cell lysates.Cell disruption can be done by any conventional method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents.

In a preferred embodiment of this invention, the mammalian expressionconstructs were transfected into CHO DXB11 cells. Stable populationswere selected using 400 μg/ml hygromycin B in HAMS/F12 medium.Expression levels were approximately 500 μg/L. To increase expressionlevels a population was selected using 100 nM methotrexate in alpha MEMmedium. The approximate expression level of this population was 5 mg/L.

The fusion construct contains the e-tag sequence at the C-terminus ofthe protein. Anti-e-tag affinity columns were purchased fromAmerican/Pharmacia Biotech. Cell culture media was filtered through a0.22 μm filter and loaded into 5 ml e-tag column at 2 ml/min. The columnwas washed with 0.2 M phosphate buffer 0.05% NaN₃, pH 7.0, and thencollected into tubes containing 0.1 volume 1M Tris buffer, pH 8.2 toneutralize the elution buffer. Alternately, the filtered culture mediumwas loaded onto a protein A column. In this case, the column was washedwith 50 mM citric acid, 300 mM NaCl, pH 6.5 and eluted with the samebuffer at pH 3.0. In both cases, the purified samples were subsequentlyloaded onto a Sephadex 200 column to separate monomer from dimer formsof the fusion protein.

Pharmaceutical Compositions:

The invention also provides pharmaceutical compositions which can beadministered to a patient to achieve a therapeutic effect.Pharmaceutical compositions of this invention can be prepared foradministration by combining fusion protein having the desired degree ofpurity and the pharmaceutically effective amount with physiologicallyacceptable carriers.

The fusion proteins of the present invention can be used inpharmaceutical compositions, for intravenous administration orsubcutaneous administration or intrathecal administration. Thus, theabove described fusion proteins preferably will be combined with anacceptable sterile pharmaceutical carrier, such as five percentdextrose, lactated Ringer's solution, normal saline, sterile water, orany other commercially prepared physiological buffer solution designedfor intravenous infusion. It will be understood that the selection ofthe carrier solution and the dosage and administration of thecomposition will vary with the subject and the particular clinicalsetting, and will be governed by standard medical procedures.

In accordance with the methods of the present invention, thesepharmaceutical compositions may be administered in amounts effective toinhibit the pathological consequences associated with excess thrombingeneration in the subject.

Administration of the fusion protein may be by a bolus intravenousinjection, by a constant intravenous infusion or by a combination ofboth routes. Alternatively, or in addition, the fusion protein mixedwith appropriate excipients may be taken into the circulation from anintramuscular site. Systemic treatment with fusion protein can bemonitored by determining the activated partial thromboplastin time (PT)on serial samples of blood taken from patient. The coagulation timeobserved in this assay is prolonged when a sufficient level of thefusion protein is achieved in the circulation.

The recombinant fusion proteins and pharmaceutical compositions of thisinvention are useful for parenteral, topical, intravenous, oral or localadministration. The pharmaceutical compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms can be administered inthe form including but not limited to tablets, capsules, powder,solutions, and emulsions.

The recombinant fusion proteins and pharmaceutical compositions of thisinvention are particularly useful for intravenous administration. Thecompositions for administration will commonly comprise a solution of thesingle chain antibody or a fusion protein comprising the single chainantibody dissolved in a pharmaceutically acceptable carrier, preferablyin an aqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. The compositions may be sterilized byconventional, well known sterilization techniques.

A typical pharmaceutical composition for intravenous administration canbe readily determined by one of ordinary skill in the art. The amountsadministered are clearly protein specific and depend on its potency andpharmacokinetic profile. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15^(th) ed., Mack PublishingCompany, Easton, Pa. (1980).

The compositions containing the present fusion proteins or a cocktailthereof (i.e., with other proteins) can be administered as therapeutictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a bleeding disorder or disease in an amountsufficient to cure or at least partially arrest the bleeding. An amountadequate to accomplish this is defined as a “therapeutically effectiveamount”. Amounts effective for this use will depend upon the severity ofthe disease and the general state of the patient's health.

Single or multiple administration of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

The fusion proteins of the invention, or their pharmaceuticallyacceptable compositions, are administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific fusion protein employed; the metabolicstability and length of action of the fusion protein; the age, bodyweight, general health, sex, and diet of the patient; the mode and timeof administration; the rate of excretion; the drug combination; theseverity of the particular disease-states; and the host undergoingtherapy. Generally, a daily therapeutically effective amount is fromabout 0.14 mg to about 14.3 mg/kg of body weight per day of a fusionprotein of the invention, or a pharmaceutically acceptable compositionthereof; preferably, from about 0.7 mg to about 10 mg/kg of body weightper day; and most preferably, from about 1.4 mg to about 7.2 mg/kg ofbody weight per day. For example, for administration to a 70 kg person,the dosage range would be from about 10 mg to about 1.0 gram per day ofa fusion protein of the invention, or a pharmaceutically acceptablecomposition thereof, preferably from about 50 mg to about 700 mg perday, and most preferably from about 100 mg to about 500 mg per day.

Gene Therapy:

A fusion protein of the invention may also be employed in accordancewith the present invention by expression of such fusion protein in vivo,which is often referred to as “gene therapy”. Thus, for example, cellsmay be engineered with a polynucleotide (DNA or RNA) encoding for thefusion protein ex vivo, the engineered cells are then provided to apatient to be treated with the fusion protein. Such methods arewell-known in the art. For example, cells may be engineered byprocedures known in the art by use of a retroviral particle containingRNA encoding for the fusion protein of the present invention.

Local delivery of the anticoagulant fusion proteins of the presentinvention using gene therapy may provide the therapeutic agent to thetarget area, the endothelial cells lining blood vessels.

Both in vitro and in vivo gene therapy methodologies are contemplated.Several methods for transferring potentially therapeutic genes todefined cell populations are known. See, e.g., Mulligan (1993) Science260:926–931. These methods include:

-   -   1) Direct gene transfer. Se, e.g., Wolff et al. (1990) Science        247: 1465–1468;    -   2) Liposome-mediated DNA transfer. See, e.g., Caplen et        al. (1995) Nature Med. 3:39–46; Crystal (1995) Nature Med.        1:15–17; Gao and Huang (1991) Biochem. Biophys. Res. Comm.        179:280–285;    -   3) Retrovirus-mediated DNA transfer. See, e.g., Kay et        al. (1993) Science 262:117–119; Anderson (1992) Science        256:808–813.    -   4) DNA Virus-mediated DNA transfer. Such DNA viruses include        adenoviruses (preferably Ad2 or Ad5 based vectors), herpes        viruses (preferably herpes simplex virus based vectors), and        parvoviruses (preferably “defective” or non-autonomous        parvovirus based vectors, more preferably adeno-associated virus        based vectors, most preferably AAV-2 based vectors). See, e.g.,        Ali et al. (1994) Gene Therapy 1:367–384; U.S. Pat. No.        4,797,368, incorporated herein by reference, and U.S. Pat. No.        5,139,941, incorporated herein by reference.

The choice of a particular vector system for transferring the gene ofinterest will depend on a variety of factors. One important factor isthe nature of the target cell population. Although retroviral vectorshave been extensively studied and used in a number of gene therapyapplications, these vectors are generally unsuited for infectingnon-dividing cells. In addition, retroviruses have the potential foroncogenicity. However, recent developments in the field of lentiviralvectors may circumvent some of these limitations. See Naldini et al.(1996) Science 272:263–267.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Adenoviruses have the advantage that they have a broad host range, caninfect quiescent or terminally differentiated cells, such as neurons orhepatocytes, and appear essentially non-oncogenic. See, e.g., Ali et al.(1994), supra, p. 367. Adenoviruses do not appear to integrate into thehost genome. Because they exist extrachromosomally, the risk ofinsertional mutagenesis is greatly reduced. Ali et al. (1994), supra, p.373.

Adeno-associated viruses exhibit similar advantages as adenoviral-basedvectors. However, AAVs exhibit site-specific integration on humanchromosome 19 (Ali et al. (1994), supra, p. 377).

In a preferred embodiment, the DNA encoding the fusion proteins of thisinvention is used in gene therapy for disorders including, but notlimited to, deep vein thrombosis, disseminated intravascularcoagulation, acute coronary syndrome or cancer with evidence ofcoagulopathy.

According to this embodiment, gene therapy with DNA encoding the fusionproteins of this invention is provided to a patient in need thereof,concurrent with, or immediately after diagnosis.

The skilled artisan will appreciate that any suitable gene therapyvector containing DNA encoding the fusion protein of the invention orDNA encoding analogs, fragments, derivatives or variants of the fusionprotein of the invention may be used in accordance with this embodiment.The techniques for constructing such a vector are known. See, e.g.,Anderson, W. F. (1998) Nature 392:25–30; Verma I. M. and Somia, N.(1998) Nature 389:239–242. Introduction of the fusion proteinDNA-containing vector to the target site may be accomplished using knowntechniques.

The gene therapy vector includes one or more promoters. Suitablepromoters which may be employed include, but are not limited to, theretroviral LTR; the SV40 promoter; and the human cytomegalovirus (CVM)promoter described in Miller et al. (1989) Biotechniques 7(9):980–990,or any other promoter (e.g., cellular promoters such as eukaryoticcellular promoters including, but not limited to, the histone, pol III,and β-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, thymidine kinase(TK) promoters, and B19 parvovirus promoters. The selection of asuitable promoter will be apparent to those skilled in the art from theteachings contained herein.

The nucleic acid sequence encoding the fusion protein of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAl promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoter.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells whichmaybe transfected include, but are not limited to, the PE501, PA317,Ψ-2, Ψ-AM, PA12, T19-14X; VT-19-17-H2, ΨCRE, ΨCRIP, GP+#-86, GP+envAm12,and DAN cell lines as described in Miller (1990) Human Gene Therapy1:5–14, which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host. The producer cellline generates infectious retroviral vector particles which include thenucleic acid sequence(s) encoding the polypeptides. Such retroviralvector particles then may be employed, to transduce eukaryotic cells,either in vitro or in vivo. The transduced eukaryotic cells will expressthe nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cellswhich may be transduced include, but are not limited to, embryonic stemcells, embryonic carcinoma cells, as well as hematopoietic stem cells,hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells,and bronchial epithelial cells.

A different approach to gene therapy is “transkaryotic therapy” whereinthe patient's cells are treated ex vivo to induce the dormantchromosomal genes to produce the protein of interest afterreintroduction to the patient. Transkaryotic therapy assumes theindividual has a normal complement of genes necessary for activation.Transkaryotic therapy involves introducing a promoter or other exogenousregulatory sequence capable of activating the nascent genes, into thechromosomal DNA of the patients' cells ex vivo, culturing and selectingfor active protein-producing cells, and then reintroducing the activatedcells into the patient with the intent that they then become fullyestablished. The “gene activated” cells then manufacture the protein ofinterest for some significant amount of time, perhaps for as long as thelife of the patient. U.S. Pat. Nos. 5,641,670 and 5,733,761 disclose indetail this concept, and are hereby incorporated by reference in theirentirety.

Kits:

This invention further relates to kits for research or diagnosticpurposes. Kits typically include one or more containers containing thesingle chain antibodies of the present invention. In a preferredembodiment, the kits comprise containers containing single chainantibodies in a form suitable for derivatizing with a second molecule,e.g., TM domain(s) or fragments thereof. In a more preferred embodimentthe kits comprise containers containing the fusion proteins of SEQ IDNO:2 or SEQ ID NO:3.

In another embodiment, the kits may contain DNA sequences encoding thefusion proteins. Preferably the DNA sequences encoding these fusionproteins are provided in a plasmid suitable for transfection into andexpression by a host cell. The plasmid may contain a promoter (often aninducible promoter) to regulate expression of the DNA in the host cell.The plasmid may also contain appropriate restriction sites to facilitatethe insertion of other DNA sequences into the plasmid to produce variousfusion proteins. The plasmids may also contain numerous other elementsto facilitate cloning and expression of the encoded proteins. Suchelements are well known to those of skill in the art and include, forexample, selectable markers, initiation codons, termination codons, andthe like.

Therapeutic Indications:

Diseases in which thrombus formation play a significant etiological roleinclude myocardial infarction, disseminated intravascular coagulation,deep vein thrombosis, pulmonary embolism, ischaemic stroke, septicshock, acute respiratory distress syndrome, unstable angina and otherarterial and venous occlusive conditions. The fusion proteins of thisinvention are useful in all of these, as well as in other diseases inwhich thrombus formation is pathological. Other pathological conditionswhere the fusion protein of this invention may be useful include cancerwith coagulopathy and inflammation. The compounds may also find use inskin and vein grafts and organ transplants. By useful it is meant thatthe compounds are useful for treatment, either to prevent disease or toprevent its progression to a more severe state. The compounds of thisinvention also provide a safe and effective anticoagulant, for example,in patients receiving bioprostheses such as heart valves. Thesecompounds may replace heparin and warfarin in the treatment of, forexample, pulmonary embolism or acute myocardial infarction. The fusionproteins of this invention may also find use in coating medical deviceswhere coagulation is an issue of concern.

Assays:

A number of laboratory assays for measuring the TM activity of a fusionprotein of the invention are available. Protein C activity can bemeasured in the assay described by Salem, H. H. et al. (1984), supra,and Galvin, J. B. et al. (1987) J. Biol. Chem. 262(5):2199–2205. Inbrief, the assay consists of two steps. The first step is the incubationof the test fusion protein with thrombin and protein C under definedconditions. In the second step, the thrombin is inactivated with hirudinor antithrombin III and heparin, and the activity of the newly activatedprotein C is determined by the used of a chromogenic substrate, wherebythe chromophore is released by the proteolytic activity of activatedprotein C. This assay is carried out with the purified reagents.

Alternately, the effect of a fusion protein can be measured using plasmaclotting time assays such as the activated partial thromboplastin time(APTT), thrombin clotting time (TCT) and/or prothrombin time (PT). Theseassays distinguish between different mechanisms of coagulationinhibition, and involve the activation of protein C. Prolongation of theclotting time in any one of these assays demonstrates that the moleculecan inhibit coagulation in plasma.

The above assays are used to identify fusion proteins with TM activitywhich are able to bind thrombin and to activate protein C in bothpurified systems and in a plasma milieu. Further assays are then used toevaluate other activities of native TM such as inhibition of thrombincatalyzed formation of fibrin from fibrinogen (Jakubowski, H. V. et al.(1986) J. Biol. Chem. 261(8): 3876–3882), inhibition of thrombinactivation of factor V (Esmon, C. T. et al. (1982). J. Biol. Chem.257(14):7944–7947), accelerated inhibition of thrombin by antithrombinIII and heparin cofactor II (Esmon, N. L. et al. (1983) J. Biol. Chem.258(20):12238–12242), inhibition of thrombin activation of factor XIII(Polgar, J. et al. (1987) Thromb. Haemost. 58(1):140), inhibition ofthrombin mediated inactivation of protein S (Thompson, E. A. and Salem,H. H. (1986) J. Clin. Inv. 78(1):13–17), and inhibition of thrombinmediated platelet activation and aggregation (Esmon, N. L. et al.(1983), supra).

The following assays, described in detail below in Example 5, are usedto measure the in vitro potency of the fusion proteins of theinvention: 1) protein C activation assay (chromogenic); 2) sTF/FVIIaactivation assay; 3) Factor X activation assay; and 4) protein Cactivation assay (on TF-rich surface).

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

The present invention will now be further described by way of thefollowing non-limiting examples. In applying the disclosure of theexample, it should be kept clearly in mind that other and differentembodiments of the methods disclosed according to the present inventionwill no doubt suggest themselves to those of skill in the relevant art.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and, all parts and percentages areby weight, unless otherwise indicated.

The entire disclosure(s) of all applications, patents and publications,cited above are hereby incorporated by reference.

The following examples are provided as a guide to assist in the practiceof the invention, and are not intended as a limitation on the scope ofthe invention.

EXAMPLE 1 The Single Chain Anti-TF Antibody Construct scFv(TF)3e10

(−18) M L G V L V L G A L A L A G L V F P E M A Q V N L R E S G G T L VQ P G G S L R L S C A A S G F S F T D A W M S W V R Q A P G K E L E W VS S I S G S G G S T Y Y A G S V K G R F T I S R D N S K N T L Y L Q M NS L R A E D T A V Y Y C A R V L S L T D Y Y W Y G M D V W G Q G T L V TV S A G G G G S G A P N F M L T Q P H S V S A S P G K T V T I S C T R SS G S V A S Y Y V Q W Y Q Q R P G S S P T T V I Y E D N H R P S G V P DR F S G S I D T S S N S A S L T I S G L K T E D E A D Y Y C Q S Y D S NN L V V F G G G T K L T V L G A A A G A P V P Y P D P L E P R A A (264)

(−18) M L G V L V L G A L A L A G L V F P E M A Q V N L R E S G G T L VQ P G G S L R L S C A A S G F S F T D A W M S W V R Q A P G K E L E W VS S I S G S G G S T Y Y A G S V K G R F T I S R D N S K N T L Y L Q M NS L R A E D T A V Y Y C A R V L S L T D Y Y W Y G M D V W G Q G T L V TV S A G G G G S G A P N F M L T Q P H S V S A S P Q R P G S S P T T V IY E D N H R P S G V P D R F S G S I D T S S N S A S L T I S G L K T E DE A D Y Y C Q S Y D S N N L V V F G G G T K L T V L G A A A G G G G S GG G G S G G G G S V E P V D P C F R A N C E Y Q C Q P L N Q T S Y L C VC A E G F A P I P G E P H R C Q L F C N Q T A C P A D C D P N T Q A S CE C P E G Y I L D D G F I C T D I D E C E N G G F C S G V C H N L P G TF E C I C G P D S A L A G Q I G T D C A A A G A P V P Y P D P L E P R AA (400)

(−18) M L G V L V L G A L A L A G L V F P E M A Q V N L R E S G G T L VQ P G G S L R L S C A A S G F S F T D A W M S W V R Q A P G K E L E W VS S I S G S G G S T Y Y A G S V K G R F T I S R D N S K N T L Y L Q M NS L R A E D T A V Y Y C A R V L S L T D Y Y W Y G M D V W G Q G T L V TV S A G G G G S N F M L T Q P H S V S A S P G K T V T I S C T R S S G SV A S Y Y V Q W Y Q Q R P G S S P T T V I Y E D N H R P S G V P D R F SG S I D T S S N S A S L T I S G L K T E D E A D Y T C Q S Y D S N N L VV F G G G T K L T V L G A A A G G G G S G G G G S G G G G S V E P V D PC F R A N C E Y Q C Q P L N Q T S Y L C V C A E G F A P I P G E P H R CQ L F C N Q T A C P A D C D P N T Q A S C E C P E G Y I L D D G F I C TD C G P D S A L A G Q I G T D C (379)

EXAMPLE 4 Expression of the Fusion Protein in Bacterial/Mammalian Cells

Bacterial expression was possible, but it yielded a protein that had amuch reduced TM cofactor activity. The fusion protein was expressed inCHO cells. The expression plasmid contains both the hygromycin B andDHFR selection markers. Original selection was done in 400 μg/mlhygromycin to select a population. The resulting population was thensubjected to 100 nM methotrexate selection. During this selection, cellsthat have amplified copies of the region of DNA containing the selectionmarker, and target gene, are selected from amongst the population. Theexpression levels were increased from approximately 0.3 mg/L to about 6mg/L as a result of this selection.

EXAMPLE 5 In Vitro Assays

1. Protein C Activation Assay (Chromogenic)

This assay was performed by mixing 20 μl each of the following proteinsin a microtiter plate: TM sample (unknown or standard), thrombin (3 nM),and protein C (1.5 μM). The assay diluent for each protein was 20 mMTris-HCl, 0.1M NaCl, 2.5 mM CaCl₂, 2.5 mg/ml BSA, pH 7.4. The wells wereincubated for 2 hours at 37° C. after which protein C activation wasterminated by the addition of 20 μl of hirudin (0.16 unit/μl 370 nM) inassay diluent and incubation for an additional 10 minutes.

The amount of activated protein C formed was detected by adding 100 μlof 1 mM S2266 (in assay diluent), and continuing to incubate the plateat 37° C. The absorbance at 405 nm in each well was read every 10seconds for 30 minutes, using a Molecular Devices plate reader. Theabsorbance data was stored, and the change in absorbance per second(slope) in each well was calculated. The change absorbance per second isproportional to pmol/ml of activated protein C.

This ratio was determined empirically using varying concentrations oftotally activated protein C. Samples containing 100% activated protein Cwere generated by mixing protein C at 0 to 1.5 μM with 60 nM rabbit TMand 30 nM thrombin, incubating for 0 to 4 hours, adding hirudin andmeasuring S2266 activity as above. Conditions under which 100% of theprotein C was activated were defined as those in which the S2266activity (A405/sec) reached plateau.

A unit of activity is defined as 1 pmole of activated protein Cgenerated per ml/min under the reagent conditions defined above.Alternatively, activity values are reported in comparison to nativedetergent solubilized rabbit TM.

2. sTF/FVIIa Activation Assay

The principle of this assay is depicted below. The tripeptidep-nitroanilide amide bond of the substrate is hydrolyzed by thesTF/FVIIa complex. The liberated chromophore product, p-nitroanilide, ismonitored at 405 nm and the concentration of product formed per unittime is calculated using a molar extinction coefficient of 9920 M⁻¹cm⁻¹.IC₅₀ values (C) are determined by fitting the initial rates into the 4parameter equation: Y=(A−D)/(1+(x/C)^B )+D

Reagents and Solutions:

-   1. Assay buffer: 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, 0.1% BSA,    pH7.5-   2. Human FVIIa (HCVIIA-0060, Haematologic Technologies Inc.):    10×working solution—prepare 20 nM solution in assay buffer prior to    use.-   3. Soluble TF (Berlex): 10×working solution—prepare 30 nM solution    in assay buffer prior to use.-   4. Chromogenic substrate S2266 (Kabi Pharmacia Hepar Inc.): Stock    solution: 10 mM in H₂O, stored at 4 C. 2.5×working solution—prepare    2.5 mM solution in assay buffer prior to use.-   5. Antibody: Prepare 2.5×dilutions in assay buffer prior to use.    Assay Conditions:

Assays are performed in a 96-well microtiter plate at room temperature.The final concentrations of the components are as follows:

sTF 3 nM Antibody vary from 1000 to 0.625 nM FVlla 2 nM S2266 1 mMAssay Procedure:

-   1. Pipette 0.1 ml of 2.5×AB (or buffer control) into each well.-   2. Add 0.025 ml 10×sTF and incubate 10 min at room temperature with    mild shaking.-   3. Add 0.025 ml 10×FVIIa, incubate 10 min at room temperature with    mild shaking.-   4. Add 0.1 ml 2.5×S2266 substrate, immediately transfer the plate    into a plate reader and measure enzyme kinetics at 405 nm at 10    seconds interval for 15 min.    3. Factor X Activation Assay:

The principle of this assay is depicted below. FVIIa is incubated withrecombinant human TF vesicles to form a protease complex capable ofactivating the substrate, FX. This complex is formed in the presence (orabsence) of different concentrations of antibody, then the substrate FXis introduced and the reaction is allowed to proceed to form theproduct, active protease FXa, which is capable of hydrolyzing thep-nitroanilide amide bond of the chromogenic substrate S2222. Theliberated chromophore product, p-nitroanilide, is monitored at 405 nmand the concentration of product formed per unit time is calculatedusing a molar extinction coefficient of 9920 M⁻¹cm⁻¹. IC₅₀ values (C)are determined by fitting the initial rates into the 4-parameterequation: Y=(A−D)/(1+(x/C)^B)+D

Reagents and Solutions:

-   1. Assay buffer: 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, 0.1% BSA,    pH7.5-   2. Human FVIIa (HCVIIA-0031, Haematologic Technologies Inc.):    4×working solution—prepare 100 pM solution in assay buffer prior to    use.-   3. Recombinant Human TF (reconstituted in our lab from Innovin,    Dade): working solution—prepare 1:480 dilution in assay buffer prior    to use.-   4. Factor X (HCX-0060, Haematologic Technologies Inc.): 4×working    solution—prepare 1000 nM solution in assay buffer prior to use.-   5. Chromogenic substrate S2222 (Kabi Pharmacia Hepar Inc.): Stock    solution: 6 mM in H₂O, stored at 4 C. Working solution—prepare 0.78    mM solution in 3.57 mM EDTA (to stop the reaction), 150 mM NaCl, 50    mM Tris-HCl pH 7.5 prior to use.-   6. Antibody: Prepare 4×dilutions in assay buffer prior to use.    Assay Conditions:    Assays are Performed in a 96-well Microtiter Plate at Room    Temperature. The Final Concentrations of the Components are as    Follows:

rTF vesicles ¼ of 1:480 dilution Antibody vary from 1000 to 0.625 nMFVlla   25 pM FX   250 nM S2222 0.546 mMAssay Procedure:

-   1. Pipette 0.015 ml of 4×AB (or buffer control) into each well.-   2. Add 0.015 ml 4×rTF vesicles.-   3. Add 0.015 ml 4×FVIIa, incubate 10 min at room temperature with    mild shaking.-   4. Add 0.015 ml 4×FX, incubate 5 min at room temperature with mild    shaking.-   5. Add 0.14 ml S2222 substrate, immediately transfer the plate into    a plate reader and measure enzyme kinetics at 405 nm at 10 seconds    interval for 15 minutes.    4. Protein C Activation Assay (on TF-rich Surface)

This assay is performed as for the chromogenic protein C activationassay listed above with the exception that in this assay humanTF-containing PC/PS vesicles are added to the fusion protein, or controlTM, before adding the thrombin and protein C.

EXAMPLE 6 Characteristics of the Anti-TF Antibodies

Seven different TF-binding antibodies were isolated from a fully humansingle chain antibody phage display library. The affinities of the sTFbinding antibodies, measured using the BIAcore, were between 35 and 470nM. The sTF/VIIa assay was used to determine if the antibodies wouldblock the formation of an active VIIa/TF complex. In the sTF/VIIa assay,binding of VIIa to sTF accelerates the rate of cleavage against thechromogenic peptide substrate S2266 by >20-fold. Antibodies that inhibitbinding of FVIIa to TF block this acceleration. From among sevendifferent antibodies isolated, only one of them, scFv(TF)3e10, did notinhibit the sTF/VIIa assay. This antibody increased the affinity ofFVIIa for sTF, decreasing the K_(D) 5-fold (FIG. 1). The K_(D) of thescFv(TF)3e10 antibody for sTF, measured using the sTF/FVIIa assay, was65.4 nM (FIG. 2). Microcalorimetry was used to compare the affinity ofscFv(TF)3e10 for TF as compared to the FVIIa/TF complex. Theseexperiments revealed that the antibody has a 20-fold higher affinity forthe TF/FVIIa complex as compared to free sTF (33 nM vs. 600 nM, FIG. 3).The antibodies were compared using the FX activation assay, whichconsists of full length TF in phospholipid vesicles, FVIIa and FX. Theamount of FXa generated is determined using the chromogenic substrateS2765. Although the scFv(TF)3e10 antibody did not have the highestaffinity as measured by BIAcore and it increased the affinity of FVIIafor sTF, it was the only antibody in the group that inhibited FXactivation and prolonged the clotting time in the PT assay. The IC₅₀ ofthe scFV(TF)3e10 (dimer) antibody for inhibition in the FX activationassay was 0.44 nM (FIG. 4) and a two-fold extension of PT occurred at417 nM (FIG. 5).

The scFv(TF)3e10 antibody was identified on the basis of binding torecombinant human soluble TF. The sequence homology of TF between thehuman and murine or human and rabbit is 58% and 71%, respectively. Theantibody binds to a unique epitope on human TF that interferes withactivation of FX by the FVIIa/TF complex. Physiologically, the antibodyhas an advantage over antibodies that compete with FVII or FVIIa bindingto TF. The K_(D) of both FVII and FVIIa in human plasma is 10 nM, orbetween 100- and 1000-fold greater than the K_(D). The off-rate for thehigh affinity complex will be slow (70 to 700 seconds, assumingk_(on)=10⁸M⁻¹sec⁻¹). In contrast, the Km of FX for the VIIa/TF complexis between 0.200 to 4 μM and the concentration of FX in human plasma is130 nM (between 0.03- and 0.65-fold K_(D)). The primary function of theFVIIa/TF complex in coagulation is to convert FX to FXa.

EXAMPLE 7 In vitro Characteristics of the Fusion ProteinscFv(TF)3e10-TMi456

The characteristics of a fusion protein of the invention,scFv(TF)3e10-TMi456, was assessed in a variety of in vitro assays. Thefusion protein, scFv(TF)3e10-TMi456, retained the ability of inhibit FXactivation by the FVIIa/TF complex (IC₅₀=0.5 nM, data not shown) andacted as a cofactor for the thrombin catalyzed activation of protein C(chromogenic assay, FIG. 6). No significant difference in TM cofactoractivity was observed between the fusion protein and Tmi456 alone in theabsence of TF-containing phospholipid vesicles. In contrast, the TMcofactor activity of the-fusion protein, but not TMi456, wasenhanced >5-fold in the presence of TF-containing phospholipid vesicles(FIG. 7). The in vitro potency of the fusion protein,scFv(TF)3e10-TMi456, against TF-induced coagulation (PT assay, extrinsiccoagulation pathway) was 6-fold better than the scFv(TF)3e10 antibodyand 17-fold better than TMi456 alone (FIG. 5). In contrast, the in vitropotency of the fusion protein against the intrinsic and commoncoagulation pathways was not significantly affected (APTT and TCTassays, data not shown). Therefore, the dose of fusion protein thatcaused a two-fold extension in the PT had only a modest effect on theAPTT, whereas TMi456, at an equivalent dose in the PT, caused a 4-foldenhancement in the APTT (FIG. 8). This in vitro profile is consistentwith that expected for TF/FVIIa-directed anticoagulants that are knownto have superior efficacy to bleeding ratios in animal models ofthrombosis. In agreement with the plasma-based coagulation assays, thefusion protein scFv(TF)3e10-TMi456 was more potent in a TF-induced wholeblood coagulation assay (Thromboelastograph, TEG) than eitherscFv(TF)3e10 or Tmi456 alone (FIG. 9). In addition, the fusion proteinscFv(TF)3e10-TMi456 had a more predictable dose response in theTF-induced whole blood coagulation assay than low molecular weightheparin (LMWH, FIG. 10). In summary, the above data demonstrate that thefusion proteins of the invention are potent and selective anticoagulantsin vitro.

EXAMPLE 8 In vivo Rat Thromboembolism Model

The TF antibody portion of the fusion protein scFv(TF)3e10-TMi456, isspecific for primate TF. A thromboembolism model triggered by human TF(thromboplastin reagent containing human recombinant TF, Ortho) wasdeveloped in conscious male Sprague-Dawley rats (350–400 g, n>7/group).In this model of disseminated intravascular coagulation (DIC), TF, viathromboplastin injection, induces pulmonary fibrin deposition,respiratory failure, and death. Equimolar doses of scFv(TF)3e10-TMi456or scFv(TF)3e10, or vehicle were injected into the tail vein followed,15 min later, by a bolus injection of thromboplastin (0.5 ml/kg). In thevehicle treated group, this dose of TF resulted in 60% lethality (LD₆₀),usually within 5 min after thromboplastin injection. The rats werescored according to the following morbidity-mortality scoring system:0=unaffected; 1=mild respiratory distress (recover within 30 min);2=severe respiratory distress (moribund, recovery required more than 60min); and 3=death. The average score was used for comparing the efficacyof the 4 different treatment groups. The results using this in vivoassay are depicted in FIG. 11. The fusion protein of the invention wasable to inhibit death and respiratory distress in this assay.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

While the invention has been illustrated with respect to the productionof certain fusion protein constructs, it is apparent that variations andmodifications of the invention can be made without departing from thespirit or scope of the invention.

1. An anticoagulant fusion protein, comprising a single chain antibodythat binds tissue factor (TF) or the factor VIIa/tissue factor(FVIIa/TF) complex operably linked to the thrombomodulin EGF456 domainand the interdomain loop between EGF3 and EGF4, wherein said fusionprotein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.2. The fusion protein of claim 1, wherein said fusion protein isglycosylated.
 3. The fusion protein of claim 1, wherein said fusionprotein is modified by the addition of polyethylene glycol.
 4. Thefusion protein of claim 1, wherein said fusion protein is biotinylatedfor binding streptavidin.
 5. A pharmaceutical composition, comprisingthe fusion protein of claim 1, which composition comprises apharmaceutically acceptable excipient and a therapeutically effectiveamount of said fusion protein.
 6. The fusion protein of claim 1, whereinsaid fusion protein can be used to form a non-thrombogenic coating onthe surface of a medical device, wherein said medical device comes incontact with blood.
 7. A kit, comprising the fusion protein of claim 1.8. An anticoagulant fusion protein, comprising a single chain antibodyof SEQ ID NO: 1 operably linked to the thrombomodulin EGF456 domain andthe interdomain loop between EGF3 and EGF4.
 9. An anticoagulant fusionprotein, comprising the Fv region of SEQ ID NO:1 operably linked to thethrombomodulin EGF456 domain and the interdomain loop between EGF3 andEGF4.